PROJECT SUMMARY More than 37 million people are living with Human Immunodeficiency Virus (HIV) infection worldwide which continues to be a global public health problem. Despite the widespread use of antiretroviral therapy (ART), up to 70% of HIV-positive individuals suffer from cognitive and behavioral deficits collectively known as HIV-associated neurocognitive disorders (HAND), for which no therapeutic options are currently available. Converging lines of evidence indicate that the HIV-1 transactivator of transcription (Tat) protein plays a crucial role in causing neurotoxicity and cognitive impairment in HAND. HIV-1 Tat exerts its neurotoxicity through interaction with crucial proteins, such as the monoamine transporters in the central nervous system (CNS). The dysregulation of dopamine (DA) neurotransmission in HAND occurs through direct interaction of Tat protein with the DA transporter (DAT) which is essential for maintaining DA homeostasis and a target of cocaine. While most ARTs cannot efficiently cross the blood-brain barrier, Tat-induced increase in DA levels accelerates viral replication in the brain. Moreover, drugs of abuse, such as cocaine, exacerbate neurological impairments. Our published work has demonstrated that Tat-induced inhibition of DAT is mediated by binding of Tat to allosteric binding site(s) on DAT, not by interacting with the DA uptake site. This provides a basis for a novel approach to address the problem by developing compounds to attenuate Tat binding to DAT by an allosteric mechanism. Our recent studies with small molecule allosteric ligands of DAT reveal that these compounds are capable of attenuating Tat-mediated effects on DAT, thus providing a potential opportunity to develop therapeutic interventions for the treatment of HAND. The research effort proposed herein is to explore the hypothesis that disruption of Tat-DAT interactions with small molecule allosteric ligands of DAT, with minimal disruption of normal DA uptake, will have therapeutic potential for prevention of neurocognitive dysfunction in HAND. The primary goal of our research is to optimize lead compounds and perform proof-of-concept pharmacological studies in animal models. To this end, the specific aims to be pursed in the proposed effort are to: (1) design and synthesize novel allosteric ligands with improved physicochemical and pharmacokinetic properties using in silico property predictions and computational docking studies with DAT-Tat complex models, (2) characterize the allosteric interaction of the compounds with human DAT in vitro to identify optimized compounds with improved physicochemical properties that can be used to alleviate Tat-induced dysfunction of DAT, and (3) determine the efficacy of selected compounds in attenuating Tat-mediated cognitive deficits and rewarding effects of cocaine in inducible Tat transgenic mice in vivo. This collaborative effort involves investigators with complementary expertise in medicinal chemistry, drug design, and biochemical and behavioral studies with the long-term goal of developing drugs for the treatment of HAND in HIV-positive patients.